Electrochemically and optically monitoring cleaving enzyme activity
A marker molecule for monitoring cleaving enzyme activity is disclosed. The marker molecule includes a protein, a peptide, or an oligonucleotide. A co-factor is conjugated to the protein, the peptide, or the oligonucleotide, thereby forming a co-factor labeled protein, a co-factor labeled peptide, or a co-factor labeled oligonucleotide. The co-factor is adapted to produce an enzymatic signal that is at least one of electrochemically and optically detectable.
This application is a continuation-in-part of U.S. application Ser. No. 11/070,568, filed Mar. 2, 2005.
BACKGROUNDThe present disclosure relates generally to electrochemically and/or optically monitoring cleaving enzyme activity, and more particularly to the electrochemical and/or optical detection of enzyme (polymerase, DNase, and protease, etc.) activity using a specially designed co-factor labeled protein, peptide, or oligonucleotide.
Genetic testing and enzyme-based assays have the potential for use in a variety of applications, ranging from genetic diagnostics of human diseases to detection of trace levels of pathogens in food products. Currently, more than 800 diseases can be diagnosed by proteomics and molecular biology analysis of nucleic acid sequences. It is likely that additional tests will be developed as further proteomic and genetic information becomes available. Protein and DNA diagnostic devices enable clinicians to efficiently detect the presence of a whole array of proteomic and genetic based diseases, including, for example, AIDS, Alzheimer's, and various forms of cancer.
The rising use of protein and/or DNA diagnostic testing devices has produced a need for low-cost, highly portable protein and/or DNA detection devices (for example, a glucometer-type “lab-on-a-chip” device) for use in various markets including health care, agriculture, food testing and bio-defense. Generally, it would be desirable that any new protein and/or DNA diagnostic devices integrate several functional analysis components within the same platform. Further, it would be desirable that such devices be reliable, inexpensive, and able to simplify the monitoring of EA (cleaving enzyme activity) and PCR (polymerase chain reaction).
SUMMARYA marker molecule for monitoring cleaving enzyme activity is disclosed. The marker molecule includes a protein, a peptide, or an oligonucleotide. A co-factor is conjugated to the protein, the peptide, or the oligonucleotide, thereby forming a co-factor labeled protein, a co-factor labeled peptide, or a co-factor labeled oligonucleotide. The co-factor is adapted to produce an enzymatic signal that is electrochemically and/or optically detectable.
A method of monitoring cleaving enzyme activity in a sample is also disclosed. The method includes exposing a co-factor labeled protein, peptide, or oligonucleotide to cleaving activity. The co-factor labeled protein, peptide, or oligonucleotide includes a protein, peptide, or oligonucleotide and a co-factor conjugated to the protein, peptide, or nucleotide. This exposure releases a fragment including the co-factor. The fragment is then combined with an apo-enzyme. Combining the fragment having the co-factor with the apo-enzyme produces an enzymatic signal that is electrochemically and/or optically detectable. The enzymatic signal, which is electrochemically and/or optically detectable, confers detection of cleaving activity.
BRIEF DESCRIPTION OF THE DRAWINGSObjects, features and advantages of embodiments of the present invention will become apparent by reference to the following detailed description and drawings, in which:
Embodiment(s) disclosed herein advantageously combine a marker molecule (e.g. a co-factor labeled protein, peptide, or nucleotide) and the production of an enzyme amplified electrochemically and/or optically detectable signal, both of which may be incorporated into a DNA diagnostic device or an EA monitoring device. This combination provides an enzyme-based electrochemical and/or optical method to detect DNA amplified via polymerase chain reaction (PCR) or to monitor cleaving enzyme activity (EA). The cleaving enzyme activity may be used to monitor the anticoagulation effect of an anticoagulation reagent (e.g. heparin), to measure activated clotting time (ACT), to measure activated partial thromboplastin time (aPTT), to measure thrombin time (TT), and/or the like. It is to be understood that embodiment(s) of the marker molecule may be integrated with, for example, a litmus paper-type strip sensing system, a multi-well plate with a plate reader, or a flow-through system with a visible spectrometer for end-point PCR detection and/or EA optical monitoring.
Referring now to
In an alternate embodiment, the marker molecule 10 is a labeled peptide or a labeled protein. Generally, these embodiments include a co-factor conjugated to a selected protein or peptide.
In the non-limitative example shown in
In embodiment(s) of the method, the co-factor (CF) 14 is adapted to produce an enzymatic signal that is electrochemically and/or optically detectable. As used herein, the term “produce” means indirectly or directly generating the enzymatic signal. In a non-limitative example, indirectly producing includes binding the co-factor (CF) 14 to an apo-enzyme to form an activated enzyme that is capable of catalyzing a reaction that results in an electrochemically and/or optically detectable enzymatic signal.
Generally, as described in more detail hereinbelow, the co-factor (CF) 14 portion of the marker molecule 10 binds with an apo-enzyme. Non-limitative examples of the co-factor (CF) 14 include prosthetic groups (organic and covalently bound to an enzyme), co-enzymes (organic and non-covalently bound to an enzyme), and metal-ion activators. Non-limitative examples of metal ion activators include iron, copper, manganese, magnesium, zinc, and the like, and combinations thereof. Specific non-limitative examples of co-factors 14 include pyrroloquinoline quinine (PQQ), flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide phosphate (NADP), or heme. Other suitable co-factors 14, specifically those that may be used in place of PQQ include, but are not limited to phenazine methosulfate, phenazine ethosulfate, naphthoquinone, derivatives thereof, benzoquinone, derivatives thereof, fluorescein, derivatives thereof, thionine, resazurin, 2,6-dichlorphenoleindophenole, orthoquinone, and derivatives thereof, and the like. It is to be understood that any fused ring compound which may be reduced by accepting 2 electrons and 2 hydrogens from a substrate (e.g. glucose) may be used as the co-factor 14. Non-limiting examples of orthoquinone (A) and its derivatives (B through I) are depicted. The derivatives have either 5- and/or 6-membered rings, 2 or 3 of which are fused together. In the non-limitative examples shown below, X═—(CH2)n, —OCH2(CH2)n, —NHCH2(CH2)n, and/or —SCH2(CH2)n, wherein n=0-6; Y═—CO2H, SO3H, SO4H, PO3H and/or PO4H; Z=CH2, NH, O, or S (with saturated bond) or CH and/or N (with unsaturated bond); and k=1-4.
The non-limitative example shown in
Referring now to
Embodiment(s) of the method integrate DNA amplification processes (non-limitative examples of which include real-time and end-point PCR) with enzymatic signal amplification. More specifically, PCR-dependent exonuclease activity can trigger enzymatic generation or amplification of a measurable electrochemical and/or optical enzymatic signal. The enzymatic signal(s) may be optically detectable via absorbance change, fluorescence, visual color change, electrochemistry, densitometry, and combinations thereof. The enzymatic signal may be electrochemically detectable via voltammetry, amperometry, coulometry, potentiometry, conductivity, tensammetry, impedance measurements, and/or combinations thereof.
Embodiment(s) of the method generally include performing a DNA amplification process on a sample 17, exposing an embodiment of the probe 10 to exonuclease activity, combining a co-factor (CF) 14 probe fragment 16 to an apo-enzyme 20, and electrochemically and/or optically detecting an enzymatic signal that results from the combination of the co-factor (CF) 14 with the apo-enzyme 20. It is to be understood that these steps may be performed substantially simultaneously or sequentially.
Non-limitative examples of the DNA amplification processes include end-point PCR, real-time PCR, PCR-free amplification (using a PCR mixture without thermocycles), rolling cycle amplification (RCA), isothermal based amplification methods, and thermocycling based amplification methods. The processes may include a reaction (or PCR) mixture and/or sample formulated such that it is compatible with desired chemistries for enzymatic signal amplification and electrochemical and/or optical detection. Such a formulation may include, but is not limited to the following: substrate(s) (a non-limitative example of which is glucose), probes 10, buffers (non-limitative examples of which include Tris, HEPES, phosphate, and the like), mediators (non-limitative examples of which include ferricyanide, ferrocene derivatives, phenazine methosulfate (PMS), Ru(III) complexes, ubiquinone (Q0), Os complexes, and the like), stabilizers (non-limitative examples of which include CaCl2, MgCl2, and the like), redox indicators (non-limitative examples of which include dichloroindolephenol (DCIP), resazurin, thionine, and the like), enzyme thermal stabilizers, barriers, oligo binders, and/or mixtures thereof.
As depicted in
Referring still to
The hydrolysis of the labeled nucleotide 10 releases (as depicted by the lightening bolt) a fragment 16 containing the co-factor (CF) 14. The co-factor (CF) 14 (e.g. a co-enzyme, prosthetic group, or metal-ion activator) of the fragment 16 may then combine with and activate an apo-enzyme 20 immobilized on the surface of a test strip 22 (a non-limitative example of which includes a PCR test strip), or multi-well plate 22, or an electrode 22. It is to be understood that the apo-enzyme 20 may also be present in solution (which may be disposed in a well of multi-well plate 22) when the assay is homogeneous. The combination of the fragment 16 and the apo-enzyme 20 forms a holo-enzyme 24, which is capable of catalyzing a reaction that converts a predetermined substrate 26 in the sample to a product 28 plus free electrons. These free electrons may reduce a mediator M(R), which is subsequently re-oxidized M(ox) by a redox indicator (In(color)) (a non-limitative example of which includes a dye) that results in the color change of the redox indicator (In(color change)). The free electrons may also reduce a co-substrate (i.e. a reactant that is transiently associated with the enzyme and becomes a product(s) that cooperates chemically with another substrate regarding formation of another product(s), a non-limitative example of which is an oxidant) with a relatively high oxidation potential (such as, for example, oxygen to hydrogen peroxide) or they may reduce the mediator M(r), which is subsequently re-oxidized M(ox) by a working electrode 22 at a lower, more selective potential.
The activation of the apo-enzyme 20 by the fragment 16 and the subsequent reaction involving the holo-enzyme 24 results in the formation (or enhancement) of an optically and/or an electrochemically measurable enzymatic signal. It is to be understood that the optical and/or electrochemical measurement of the enzymatic signal corresponds to a measurement of the target DNA 17. The optical detection of GBS cfb gene using an embodiment of the method shown in
In this non-limitative example, a 0.2 ml PCR tube, about 3 μl (3.3*10+5) of GBS genomic DNA (ATCC BAA-611D), about 1 μl of 100 μM p-probe (a PQQ-probe), and about 1.0 μl of 100 μM primers were added to ingredients of the PCR mix (about 220 μM dNTP, about 1.65 mM MgCl2, about 22 U Taq Polymerase/ml, about 55 mM KCl, and 22 mM Tris-HCl (pH 8.4), and stabilizers) to render a total reaction volume of about 50 μL. A hybridization primer-probe assay targeting the cfb gene that encodes the CAMP-factor protein was used. The thermal cycle run profile for the GBS cfb gene consists of a hot start of 1 cycle at about 94° C. for about 120 seconds, followed by amplification which includes 35 cycles at about 94° C. for about 15 seconds (i.e., denaturation occurs), exposure to about 55° C. for about 30 seconds for annealing, and exposure to about 72° C. for about 30 seconds for extension to occur. 1 cooling cycle was performed at about 68° C. for about 7 minutes. The PCR results were validated using conventional gel-electrophoresis method to identify the corresponding amplicons.
Post-PCR amplicons were transferred to a 96-well plate, to which assay reagents (about 30 μl 0.5 mM DCPIP, about 7.5 μL 20 mM CaCl2, about 25 μL 80 mM glucose, and about 5 μL 57 μg/mL GDH) were added for the optical detection based upon apo-GDH-based signal amplification. DCPIP was used as a color indicator (λmax=605 nm), which was reduced by the reconstituted holo-GDH. The absorbance was measured at 590 nm.
In
Referring back to
Referring back to the DNA amplification processes, in an embodiment using a PCR-free amplification process, the DNA amplification occurs without thermal denaturation of double stranded (ds) template DNA. By definition, the melting temperature of a given sample of DNA means the temperature at which half the population of dsDNA in the sample exists denatured, i.e. in a single-stranded form. This temperature is the inflection point of a sigmoidal melting curve of the given DNA sample. As such, a slight portion of dsDNA can be denatured and exist in a single stranded form at temperatures other than the melting temperature. In the presence of primers and probes 10 with appropriate sequences, the polymerase- and exonuclease-based amplification reaction may still occur at a relatively slow rate. In a non-limitative example embodiment, while the number of amplicons and PQQ released increases proportionally to the natural progression of the polymerase- and exonuclease-based amplification at room temperature (or at/near the melting temperature of primers and PQQ-probes), the activation of the enzyme (e.g. GDH) is highly pronounced, in part, because the product of this amplification reaction is theoretically capable of activating a single molecule (e.g. GDH) whose turnover number is around 10,000. Therefore, even without the thermocycling, an electrochemical or optical signal change indicating the existence of a gene sequence of interest may be generated.
In an embodiment using an end-point detection DNA amplification process, holo-enzyme 24 activity (which generates the enzymatic signal) is measured before and after the entire process. The methods for detection include, but are not limited to visual color change, absorbance change, fluorometry, electrochemistry, densitometry, voltammetry, amperometry, coulometry, potentiometry, conductivity, tensammetry, impedance measurements, and/or the like. It is to be understood that a sample designation as to whether “positive (with target DNA)” or “negative (without target DNA)” for a given DNA sequence may depend, at least in part, on a predetermined criterion involving the magnitude of the change in optical and/or electrochemical signal observed before and after the amplification process. In one embodiment using end-point detection, the PCR and detection processes may be performed sequentially as two separate steps in two separate and/or different spatial environments. In another embodiment using end-point detection, the PCR and optical and/or electrochemical detection may be batch processed in one integrated step in the same spatial environment. In a non-limitative example using batch processing, thermophilic or thermally stabilized enzymes (non-limitative examples of which include sol-gel and probe encapsulated by biologically localized embedding (PEBBLE)) may be used.
In an embodiment using a real-time detection DNA amplification process, holo-enzyme 24 activity (which generates the enzymatic signal) is measured continuously, or in many closely spaced (in time) discrete measurements, throughout the entire process. The optical and/or electrochemical signal may be detected using the methods previously described under oxidative or reductive conditions. It is to be understood that a sample designation as to whether “positive (with target DNA)” or “negative (without target DNA)” for a given DNA sequence may depend, at least in part, on a predetermined criterion involving the magnitude of the Delta comparing signal measurements before and after the PCR for each thermal cycle. In one embodiment using real-time detection, the PCR and detection processes may be performed sequentially as two separate steps in two separate and/or different spatial environments. In another embodiment using real-time detection, the PCR and optical and/or electrochemical detection may be batch processed in one integrated step in the same spatial environment. In a non-limitative example using batch processing, thermophilic or thermally stabilized enzymes (non-limitative examples of which include sol-gel and PEBBLE) may be used.
Referring now to
In the non-limitative example shown in
Referring now to
In the non-limitative example shown in
This product may be subsequently purified using a reverse phase HPLC column. The non-limitative example the PQQ-peptide conjugate (marker molecule 10) shown in
Referring now to
Embodiment(s) of the method generally include exposing an embodiment of the co-factor labeled protein, peptide, or oligonucleotide 10 to cleaving activity (e.g. peptide, protein, or nucleotide cleaving activity), combining a co-factor (CF) 14 fragment 16 to an apo-enzyme 20, and optically or electrochemically detecting an enzymatic signal that results from the combination of the fragment 16 with an apo-enzyme 20. It is to be understood that these steps may be performed substantially simultaneously or sequentially.
Cleaving enzyme activity processes may include formulating a sample such that it is compatible with desired chemistries for enzymatic signal amplification and optical and/or electrochemical detection. Such a formulation may include, but is not limited to the following: substrate(s) (a non-limitative example of which is glucose), marker molecules 10 (e.g. PQQ-protamine conjugate(s)), buffers (non-limitative examples of which include Tris, HEPES, phosphate, and the like), mediators (non-limitative examples of which include ferricyanide, ferrocene derivatives, phenazine methosulfate (PMS), Ru(III) complexes, ubiquinone (Qo), Os complexes, and the like), stabilizers (non-limitative examples of which include CaCl2, MgCl2, and the like), redox inhibitors (dichloroindolephenol (DCIP), resazurin, thionine, and the like), enzyme thermal stabilizers, barriers, oligo binders, and/or mixtures thereof.
As depicted in
The hydrolysis of the marker molecule 10 by peptide cleaving enzyme (e.g., trypsin) releases (as depicted by the lightening bolt) a fragment 16 containing the co-factor (CF) 14. The fragment 16 containing the co-factor (CF) 14 may then combine with and activate an apo-enzyme 20 immobilized on the surface of a test strip 22 or a working electrode. It is to be understood that the apo-enzyme 20 may also be present in solution when the assay is homogeneous. The combination of the fragment 16 and the apo-enzyme 20 forms a holo-enzyme 24, which is capable of catalyzing a reaction that converts a predetermined substrate 26 in the sample to a product 28 plus free electrons. These free electrons may reduce a mediator M(R), which is subsequently re-oxidized M(OX) by a redox indicator (In(color)) (a non-limitative example of which includes a dye) that results in the color change of the redox indicator (In(color change)). The free electrons may also reduce a co-substrate with a relatively high oxidation potential (such as, for example, oxygen to hydrogen peroxide) or they may reduce the mediator M(r), which is subsequently re-oxidized M(ox) by a working electrode at a lower, more selective potential.
The activation of the apo-enzyme 20 by the fragment 16 and the subsequent reaction involving the holo-enzyme 24 results in the formation or amplification of an optically and/or electrochemically measurable enzymatic signal. It is to be understood that the optical and/or electrochemical measurement of the enzymatic signal corresponds to monitoring cleaving enzyme activity.
The embodiment shown in
Referring back to the cleaving processes, holo-enzyme 24 activity (which generates the enzymatic signal) is measured through the entire process in real time. The methods for detection include, but are not limited to visual color change, absorbance change, fluorometry, electrochemistry (potentiometry, amperometry, voltametry, etc.), and the like. In a non-limitative example using batch processing, thermophilic or thermally stabilized enzymes (non-limitative examples of which include sol-gel and probe encapsulated by biologically localized embedding (PEBBLE)) may be used.
It is to be understood that the embodiments of the probe 10 disclosed herein may be chimeric probes including a linker molecule positioned between the co-factor 14 and the peptide, protein, or oligonucleotide. Non-limitative examples of such linker molecules include PQQ-peptide-oligonucleotide probe, PQQ-peptide-PNA oligomer probe, and PQQ-peptide-DNA oligomer probe. It is to be understood that the chimeric probe may recognize the target DNA sequence and may release the probe fragment 16 by protease action on the linker molecule. The fragment 16 activates the apo-enzyme 20 to produce a signal change capable of assaying the target DNA sequence qualitatively and/or quantitatively. It is to be further understood that the linker molecule may be any molecule capable of being cleaved by a chemical or biological reaction, such that the probe fragment 16 is generated to activate the apo-enzyme 20. In a non-limitative example, the linker is a peptide.
Experimental
Homogeneous Optical and Electrochemical Assay for Trypsin activity with GDH and PQQ-Protamine Marker Molecule
The PQQ-protamine marker molecule (4 nM) solutions included APO-GDH (1 μM), 0.2mM CaCl2, and 40 mM Glucose. 60 μM PMS and a redox indicator (0.3 mM DCIP) were also added. (See
Homogeneous Optical and Electrochemical Assay for Trypsin activity with GDH and PQQ-Peptide Marker Molecule
The PQQ-peptide marker molecule (2 nM) solutions included APO-GDH (2 nM), 0.2mM CaCl2, and 40 mM Glucose. 60 μM PMS and a redox indicator (0.3 mM DCIP) were also added. (See
Homogeneous Optical and Electrochemical Assay for DNA with GDH and PQQ (See
The PQQ solutions (variable concentrations) included APO-GDH (1 μM), 2 mM CaCl2, and 40 mM Glucose. 50 μM PMS and a redox indicator (0.1-0.2 mM DCIP, 0.025 mM resazurin, or 0.05 mM thionine) were also added. PQQ at 0.1 nM was detectable.
Homogeneous Optical and Electrochemical Assay for DNase activity with PQQ-Oligonucleotide Marker Molecule
The PQQ-oligonucleotide marker molecule (10 nM) solutions included APO-GDH (1 μM), 0.2 mM CaCl2, and 40 mM Glucose. 60 μM PMS and a redox indicator (0.3 mM DCIP) were also added. (See
While several embodiments have been described in detail, it will be apparent to those skilled in the art that the disclosed embodiments may be modified. Therefore, the foregoing description is to be considered exemplary rather than limiting.
Claims
1. A marker molecule for monitoring cleaving enzyme activity, the marker molecule comprising:
- a protein, a peptide, or an oligonucleotide; and
- a co-factor conjugated to the protein, the peptide, or the oligonucleotide, thereby forming a co-factor labeled protein, a co-factor labeled peptide, or a co-factor labeled oligonucleotide;
- wherein the co-factor is adapted to produce an enzymatic signal that is at least one of electrochemically and optically detectable.
2. The marker molecule as defined in claim 1 wherein the co-factor is one of a prosthetic group, a co-enzyme, and a metal-ion activator.
3. The marker molecule as defined in claim 1 wherein the co-factor is one of pyrroloquinoline quinone, flavin adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, heme, phenazine methosulfate, phenazine ethosulfate, naphthoquinone, derivatives thereof, benzoquinone, derivatives thereof, fluorescein, derivatives thereof, thionine, resazurin, 2,6-dichlorphenoleindophenole, orthoquinone, and derivatives thereof.
4. The marker molecule as defined in claim 1 wherein the enzymatic signal is optically detectable via absorbance change, fluorescence, visual color change, electrochemistry, densitometry, and combinations thereof.
5. The marker molecule as defined in claim 1 wherein the enzymatic signal is electrochemically detectable via at least one of voltammetry, amperometry, coulometry, potentiometry, conductivity, tensammetry, impedance measurements, or combinations thereof.
6. The marker molecule as defined in claim 1 wherein the co-factor is adapted to release from the labeled protein, the labeled peptide, or the labeled oligonucleotide and to bind to and activate an apo-enzyme, thereby forming a holo-enzyme that is adapted to catalyze a reaction to produce electrons.
7. The marker molecule as defined in claim 1 wherein the co-factor labeled protein, the co-factor labeled peptide, or the co-factor labeled oligonucleotide further comprises a linker molecule between the co-factor and the protein, the peptide, or the oligonucleotide.
8. A labeled oligonucleotide for enzyme amplified target DNA detection, the labeled oligonucleotide comprising:
- a site-specific sequence; and
- a co-factor conjugated to the site-specific sequence;
- wherein the co-factor is adapted to produce an enzymatic signal that is at least one of electrochemically and optically detectable.
9. The labeled oligonucleotide as defined in claim 8 wherein the co-factor is one of a prosthetic group, a co-enzyme, and a metal-ion activator.
10. The labeled oligonucleotide as defined in claim 8 wherein the co-factor is one of pyrroloquinoline quinone, flavin adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, heme, phenazine methosulfate, phenazine ethosulfate, naphthoquinone, derivatives thereof, benzoquinone, derivatives thereof, fluorescein, derivatives thereof, thionine, resazurin, 2,6-dichlorphenoleindophenole, orthoquinone, and derivatives thereof.
11. The labeled oligonucleotide as defined in claim 8 wherein the site specific sequence is 5′-amino-X—PO4-CCAAAAGGTACACCTGTTTGAG-3′, with X selected from —(CH2)n—, —(CH2)oO(CH2)p—, and —(CH2)qS(CH2)r—, with n selected from the numbers two through twelve, and with o, p, q, and r selected from the numbers two through six, wherein the co-factor is pyrroloquinoline quinone, and wherein the target DNA is from P. pachyrhizi.
12. The labeled oligonucleotide as defined in claim 8 wherein the enzymatic signal is optically detectable via absorbance change, fluorescence, visual color change, electrochemistry, densitometry, and combinations thereof.
13. The labeled oligonucleotide as defined in claim 8 wherein the enzymatic signal is electrochemically detectable via at least one of voltammetry, amperometry, coulometry, potentiometry, conductivity, tensammetry, impedance measurements, or combinations thereof.
14. The labeled oligonucleotide as defined in claim 8 wherein the co-factor is adapted to release from the site-specific sequence and to bind to and activate an apo-enzyme, thereby forming a holo-enzyme that is adapted to catalyze a reaction to produce electrons.
15. The labeled oligonucleotide as defined in claim 1, further comprising a linker attaching the co-factor to the protein, the peptide, or the oligonucleotide.
16. A method of detecting target DNA in a sample, the method comprising:
- performing a DNA amplification process on the sample;
- exposing a labeled oligonucleotide to exonuclease activity, the labeled oligonucleotide including a site-specific sequence and a co-factor conjugated to the site-specific sequence, the exposing thereby releasing a fragment including the co-factor;
- combining the fragment with an apo-enzyme, wherein combining the fragment including the co-factor with the apo-enzyme produces an enzymatic signal that is at least one of electrochemically and optically detectable; and
- at least one of electrochemically and optically detecting the enzymatic signal, thereby detecting the target DNA.
17. The method as defined in claim 16 wherein the DNA amplification process is at least one of real time PCR, end-point PCR, PCR-free amplification, rolling cycle amplification, isothermal based amplification methods, thermocycling based amplification methods, or combinations thereof.
18. The method as defined in claim 17 wherein the DNA amplification process is real time PCR and wherein the enzymatic signal is detected at predetermined intervals during the amplification process.
19. The method as defined in claim 17 wherein the DNA amplification process is end-point PCR and wherein the enzymatic signal is detected prior to and after the amplification process.
20. The method as defined in claim 17 wherein the DNA amplification process is PCR-free amplification, and wherein the enzymatic signal is detected at least one of prior to, at predetermined intervals during, or after the amplification process.
21. The method as defined in claim 16 wherein the DNA amplification process includes exposing the sample to a PCR mixture including at least one of substrates, probes, buffers, mediators, stabilizers, redox indicators, calcium chloride, magnesium chloride, or combinations thereof.
22. The method as defined in claim 16 wherein the performing, exposing, combining, and detecting occur one of substantially simultaneously and sequentially.
23. The method as defined in claim 16 wherein optically detecting the enzymatic signal is accomplished via absorbance change, fluorescence, visual color change, electrochemistry, densitometry, and combinations thereof.
24. The method as defined in claim 16 wherein electrochemically detecting the enzymatic signal is accomplished via at least one of voltammetry, amperometry, coulometry, potentiometry, conductivity, tensammetry, impedance measurements, or combinations thereof.
25. A method of monitoring cleaving enzyme activity in a sample, the method comprising:
- exposing a co-factor labeled protein, peptide, or oligonucleotide to cleaving activity, the co-factor labeled protein, peptide, or nucleotide including a protein, peptide, or nucloetide and a co-factor conjugated to the protein, peptide, or nucleotide, the exposing thereby releasing a fragment including the co-factor;
- combining the fragment with an apo-enzyme, wherein combining the fragment including the co-factor with the apo-enzyme produces an enzymatic signal that is at least one of electrochemically and optically detectable; and
- at least one of electrochemically and optically detecting the enzymatic signal, thereby monitoring cleaving enzyme activity.
26. The method as defined in claim 25 wherein optically detecting the enzymatic signal may be accomplished via at least one of absorbance change, fluorescence, visual color change, electrochemistry, densitometry, or combinations thereof.
27. The method as defined in claim 25 wherein electrochemically detecting the enzymatic signal is accomplished via at least one of voltammetry, amperometry, coulometry, potentiometry, conductivity, tensammetry, impedance measurements, or combinations thereof.
28. The method as defined in claim 25 wherein the co-factor is one of a prosthetic group, a co-enzyme, or a metal-ion activator.
29. The method as defined in claim 28 wherein the co-factor is one of pyrroloquinoline quinone, flavin adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, heme, phenazine methosulfate, phenazine ethosulfate, naphthoquinone, derivatives thereof, benzoquinone, derivatives thereof, fluorescein, derivatives thereof, thionine, resazurin, 2,6-dichlorphenoleindophenole, orthoquinone, and derivatives thereof.
30. The method as defined in claim 25 wherein the cleaving activity is peptide cleaving activity, nucleotide cleaving activity, or protein cleaving activity.
31. The method as defined in claim 25 wherein the cleaving activity is accomplished via a cleaving enzyme.
32. The method as defined in claim 31 wherein the cleaving enzyme is selected from DNases, polymerases, proteases, renin, FVa, FXa, FIIa, and HIV-protease.
33. The method as defined in claim 25 wherein the cleaving enzyme activity is adapted to monitor an anticoagulation effect of an anticoagulation reagent, to measure activated clotting time, to measure activated partial thromboplastic time, to measure thrombin time, or combinations thereof.
34. An optical diagnostic device for detecting target DNA, the device comprising:
- a multi-well plate having a microplate reader; and
- a reaction mixture disposed within the multi-well plate, the reaction mixture including an apo-enzyme and a labeled oligonucleotide, the labeled oligonucleotide including:
- a site-specific sequence; and
- a co-factor conjugated to the site-specific sequence;
- wherein the co-factor is cleaveable from the labeled oligonucleotide to form a probe fragment that binds to the apo-enzyme to produce an enzymatic signal that is optically detectable.
35. The optical diagnostic device as defined in claim 34 wherein the reaction mixture further includes at least one of enzyme substrates, buffers, mediators, stabilizers, redox indicators, color indicators, calcium chloride, magnesium chloride, or combinations thereof.
36. The optical diagnostic device as defined in claim 34 wherein the enzymatic signal is optically detectable via absorbance change, fluorescence, visual color change, electrochemistry, densitometry, and combinations thereof.
37. An optical diagnostic device for optically monitoring cleaving enzyme activity, the device comprising:
- a multi-well plate having a microplate reader; and
- a reaction mixture disposed within the multi-well plate, the reaction mixture including an apo-enzyme and a marker molecule, the marker molecule including: a protein, a peptide, or an oligonucleotide; and a co-factor conjugated to the protein, the peptide, or the oligonucleotide, thereby forming a co-factor labeled protein, a co-factor labeled peptide, or a co-factor labeled oligonucleotide;
- wherein the co-factor is cleaveable from the protein, the peptide, or the oligonucleotide and is able to bind to and activate the apo-enzyme to produce an enzymatic signal that is optically detectable.
38. The optical diagnostic device as defined in claim 37 wherein the reaction mixture further includes at least one of enzyme substrates, buffers, mediators, stabilizers, redox indicators, color indicators, calcium chloride, magnesium chloride, or combinations thereof.
39. The optical diagnostic device as defined in claim 37 wherein the enzymatic signal is optically detectable via absorbance change, fluorescence, visual color change, electrochemistry, densitometry, and combinations thereof.
40. An electrochemical diagnostic device for detecting target DNA, the device comprising:
- at least one electrode having an apo-enzyme immobilized thereon; and
- a labeled oligonucleotide in electrochemical contact with the at least one electrode, the labeled oligonucleotide including: a site-specific sequence; and a co-factor conjugated to the site-specific sequence;
- wherein the co-factor is cleaveable from the labeled oligonucleotide to form a probe fragment that binds to the apo-enzyme to produce an enzymatic signal that is electrochemically detectable.
41. The electrochemical diagnostic device as defined in claim 40 wherein the enzymatic signal is electrochemically detectable via at least one of voltammetry, amperometry, coulometry, potentiometry, conductivity, tensammetry, impedance measurements, or combinations thereof.
42. An electrochemical diagnostic device for electrochemically monitoring cleaving enzyme activity, the device comprising:
- at least one electrode having an apo-enzyme immobilized thereon; and
- a marker molecule in electrochemical contact with the at least one electrode, the marker molecule including: a protein, a peptide, or an oligonucleotide; and a co-factor conjugated to the protein, the peptide, or the oligonucleotide,
- wherein the co-factor is cleaveable from the protein, the peptide, or the oligonucleotide and is able to bind to and activate the apo-enzyme to produce an enzymatic signal that is electrochemically detectable.
43. The electrochemical diagnostic device as defined in claim 42 wherein the enzymatic signal is electrochemically detectable via at least one of voltammetry, amperometry, coulometry, potentiometry, conductivity, tensammetry, impedance measurements, or combinations thereof.
44. A labeled oligonucleotide for enzyme amplified target DNA detection, the labeled oligonucleotide comprising:
- a site-specific sequence;
- a co-factor; and
- a linker conjugating the co-factor to the site-specific sequence;
- wherein the linker is cleaveable, thereby forming a co-factor fragment that is adapted to produce an enzymatic signal that is at least one of electrochemically and optically detectable.
Type: Application
Filed: Nov 1, 2005
Publication Date: Sep 7, 2006
Inventors: Hyoungsik Yim (Ann Arbor, MI), Mark Meyerhoff (Ann Arbor, MI), Dongxuan Shen (Ann Arbor, MI), Sangyeul Hwang (Ann Arbor, MI)
Application Number: 11/264,288
International Classification: C12Q 1/37 (20060101); C07K 14/47 (20060101); C07K 14/80 (20060101);